1. Field of the Invention
The present invention relates to the field of inflammation caused by injury or disease. More specifically, the invention relates to inflammatory mediators and their antagonists.
2. Description of Related Art
Uncontrolled inflammatory processes, whether initiated by infections, immunologic, or environmental factors, represent extremely explosive and potentially destructive biologic responses. Central to these inflammatory processes are the recruitment and activation of leukocytes. This activation can occur both locally within tissues, as well as systemically, e.g. as in cytokine storms. In general, a cytokine storm is induced by a viral infection (e.g., influenza flu), by a gram-negative bacterial infection (endotoxin), or in patients infected by a virus and then infected by a gram-negative bacterial infection due to a compromised immune system.
Cytokine storms result in profound local and systemic activation of the immune system and can lead to organ failure and death. A cytokine storm is the systemic expression of a healthy and vigorous immune system resulting in the release of many inflammatory mediators, including chemokines, cytokines, oxygen free radicals, digestive enzymes, and coagulation factors. Key pro-inflammatory mediators in the cytokine storm are Tumor Necrosis Factor α (TNF-α), Interleukin 1β(IL-1β), Interleukin 8 (IL-8), and Interleukin 6 (IL-6). Acute respiratory viral infection results in a cytokine storm affecting the lungs, and subsequently damages the alveoli and lung tissue. Activated immune cells release toxic substances such as free radicals and digestive enzymes, resulting in tissue necrosis and organ failure. In the absence of prompt medical intervention to stop the cytokine storm, the lung will suffer permanent damage. Deaths will usually result from multisystem organ failure and not from lung failure. Avian influenza is known to affect more organs (including the GI tract) than does ordinary influenza, which affects primarily the lung.
The Swine influenza H1N1 virus is now a prominent flu strain for which a vaccine is being tested. In the 1997 outbreak, elevated blood levels of Interleukin 6 (IL-6) and Tumor Necrosis Factor α (TNF-α), interferon-γ, and soluble interleukin-2 receptor were observed in influenza patients. In the 2003 outbreak, elevated levels of the chemokines interferon-induced protein 10, monocyte chemoattractant protein 1, and monokine induced by interferon-γ were found in infected patients three to eight days after the onset of illness. Clinical studies suggest that the innate immune responses to Avian influenza A (H5N1) may contribute to disease pathogenesis. In general, the levels of inflammatory mediators were found to be higher among patients who died than among those who survived.
There is growing concern that Avian influenza (bird flu) may again spread across the world in a pandemic that far surpasses the 1918 pandemic. Despite the threat that influenza represents, relatively little is known about the mediators and mechanisms that drive the progression of this disease, particularly the early mediators that are associated with pulmonary inflammation and loss of lung function. Central to this process is the recruitment and activation of leukocytes in the virus-infected lung. Although leukocytes, such as neutrophils, monocyte/macrophages, and lymphocytes, are generally thought to play a protective role during early infections in the form of inflammation, this inflammation when uncontrolled has the potential to totally destroy healthy tissue, including the lungs. Additionally, recruited leukocytes, such as the macrophages, can be co-opted by the influenza virus as a source/viral host for additional virus production. The Avian and Swine influenza usually begins much like more prevalent yearly influenza-fever, cough, sore throat and muscle aches, but the virus forces the immune system into overload causing organ failures and in some cases death. It is still not clear what the endogenous inflammatory mediators are that modulate the initial inflammatory responses (innate immunity) during influenza infection. Even less is known about the early immune cascade of events that tilt the balance to either immune protection with resolution and recovery or to the devastating overactive inflammatory response leading to immunopathology disease and death.
Previous studies have identified some virus-induced chemotactic factors that are associated with later stages of influenza infection, i.e. >48 hrs (e.g. Interleukin-8 (IL-8), etc.). However, no one has identified early chemotactic factors that are responsible for the initial recruitment of leukocytes seen during early stages of influenza infections in the lungs.
Clinically, vaccines and anti-viral medications are the two most common approaches generally used to prevent and treat viral infections. However, neither can control the excessive host inflammatory response, including cytokine storms, which occur secondary to viral influenza infections and can cause organ failure and death. In addition, vaccine shortages are common due to insufficient sources of viral coat materials and the cost of stockpiling enough vaccine for an entire population. Another disadvantage for both vaccinations and anti-viral medications is that viral mutations can render the drug or vaccine ineffective.
Early diagnosis in the form of a quick point-of-care test is a vital element in the defense against Swine and Avian influenza. Vaccination, drug treatment, and containment are all under consideration for Swine and Avian influenza preparedness, but their use cannot be optimized unless infection is quickly detected. Early stages of Swine and Avian flu influenza, when transmission first begins, lack distinguishing clinical symptoms from seasonal influenza and thus require a sensitive biochemical test. Because such a test will most likely be used under diverse conditions, ranging, for example, from emergency rooms to airports, it needs to be as straightforward and robust as possible.
The power of containment is still the traditional first line of defense against an epidemic, but rapid identification of infections in individuals or animals is crucial to treatment and to containment strategies. Unfortunately, current detection technologies, for example, PCR (polymerase chain reaction), viral culture, and immunoassays, do not provide a quick and easy test. PCR, which analyzes the viral genome, is the most sensitive but is slow (minimum time, two hours), requires highly trained personnel, and can miss new viral strains. Viral culture is the gold standard for diagnosis but is even slower (minimum time, several days), is more difficult to perform than PCR, and requires special high-security labs to minimize the risk of release of virions that are formed during the test. Immunoassays, like those used for the familiar home pregnancy test, give rapid results and are easy to perform but currently lack the necessary sensitivity and specificity to distinguish Swine and Avian from seasonal influenza reliably. The few such immunoassay-based tests that claim to detect Swine and Avian influenza are purportedly insensitive and are thus unlikely to pick up newly evolving strains.
In addition to viral infections, cytokine storms can also occur with bacterial infections. Sepsis is a severe systemic inflammatory response and is one example of a pathologic condition associated with cytokine storms. Sepsis is an often lethal hemodynamic collapse, which is usually the result of a super infection by gram-negative bacteria producing endotoxins. Sepsis is also classified as Septic Shock Syndrome (SSS) and is the number one cause of death in hospitals. Although a wide variety of microorganisms can cause sepsis, one of the major causes of bacterial sepsis is the Gram positive Staphylococcus aureus (S. aureus). Traditionally, methicillin-resistant S. aureus (MRSA) infections have been known to cause sepsis in hospitals and have been limited to immunocompromised patients or individuals with predisposing risk factors. Extensive use of antibiotics and increased numbers of drug-resistant pathogens increases the risk of sepsis in the rest of the population. Recently, there has been an alarming epidemic caused by community-acquired MRSA (CA-MRSA), which can cause severe infections that result in overactive host inflammatory response, organ failure, and even death in otherwise healthy individuals. Current treatment modalities for hospitals and CA-MRSA are of limited utility and success. Formyl peptides such as the phenol-soluble modulin 3a peptide (PSM3a) are key bacterial products secreted by S. aureus with leukocyte chemotactic activity (i.e., leukocyte recruitment) for phagocytic leukocytes.
Uncontrolled inflammatory processes initiated by bacteria-induced sepsis represent extremely explosive and potentially destructive biologic responses. Central to these inflammatory processes are the recruitment and activation of leukocytes that lead to tissue injury and organ failure. This activation can occur both locally within tissues, as well as systemically, e.g. cytokine storms. Some of the major pro-inflammatory cytokines involved in septic shock are Interleukin 1β(IL-1β), Interleukin 6 (IL-6), Interleukin 8 (IL-8), Interleukin 18 (IL-18), and Tumor Necrosis Factor alpha (TNF-a). These cytokine storm mediators are associated with tachycardia, hypotension, procoagulatory activities, as well as leukocyte recruitment, adhesion, and activation, which further contribute to the destruction and dysfunction of many organs. A number of studies have shown that elevated levels of these cytokines correlated with poor patient outcome.
Leukocyte chemotactic factors are potent activators/mediators of leukocytes in vitro and in vivo. Leukocyte chemotactic factors (LCF) can induce a variety of activities in leukocytes, including recruiting leukocytes from the circulation into sites of infection or tissue injury, stimulating the secretion of adhesion molecules by leukocytes and vascular endothelial cells and accordingly increasing the adhesion of cells to the sites of infection and injury, and activating leukocytes and vascular endothelial cells to release chemokines, cytokines, and toxic agents such as oxygen metabolites and digestive enzymes.
The Nourin family is comprised of unique tissue-derived inflammatory mediators released by local tissues in response to diverse types of injury and infections. These mediators are potent attractants for leukocytes and appear to be among the first compounds released by injured tissues. As an early inflammatory signal, the tissue-derived factors not only initiate the cascade of events leading to inflammation, but also amplify the response. These factors activate immune cells to release a number of cytokines, chemokines, oxidants, and proteolytic enzymes and they exhibit their activities as low and high molecular weight proteins. Specifically, Nourin stimulates neutrophils and monocytes to release adhesion molecules, chemokines, and cytokines such as LECAM, IL-8, IL-1β, and TNF-α, as well as oxidants and digestive enzymes. However, the extent of the role of Nourin family peptides in inflammation due to infection has not yet been determined.
Structurally, Nourin is a 3 KDa host-derived formyl peptide and it belongs to a family of N-formyl methionyl peptides, with a common motif of formyl-methionyl at the N-terminus. Upon cell injury (trauma) or infection-induced cell damage, Nourin released from the damaged cells binds to the formyl peptide receptor (FPR) on leukocytes and subsequently induces activation of neutrophils and monocytes. It then acts as a chemotactic factor, guiding the migration of these cells to the inflammatory site. Because Nourin is released by local tissues following injury and infections and it contributes to the induction of an inflammatory response, it can be characterized as an Alarmin, a new terminology for endogenous factors, which signal to the immune system the presence of tissue damage. Because Nourin stimulates the release of IL-8, as well other chemokines and cytokines by neutrophils and monocytes, it plays a key role not only in the initiation of tissue inflammation such as in the lung after infection, but also in the amplification of circulating inflammatory response, which leads to cytokines dysfunction (cytokine storm), organ failure, and death.
Formyl peptides are not only released by host cells, but can also be bacterial products. For example, formyl peptides are key bacterial products secreted by S. aureus with leukocyte chemotactic activity (i.e. leukocyte recruitment) for phagocytic leukocytes. Therefore, general inhibitors of formyl peptides should inhibit both the host response of patients with sepsis, for example, and should also inhibit the pro-inflammatory formylated peptides released from bacteria.
The inventors have recognized that there exists an urgent need in the art for substances that can control the general inflammatory response when induced by injury or disease, such as viral or bacterial infections. The development of drugs that can combat both viral cytokine storms and can also be used to control sepsis is also recognized by the inventors as needed in the field. In addition, the inventors have recognized that there also exists a need for diagnostic methods for early detection of diseases or disorders involving inflammatory pathways.